We have recently developed a preconditioning regimen of plasmapheresis (PP) and CMV hyperimmune globulin (CMVIg) that, in combination with standard immunosuppressive medications, produces durable suppression of antibodies to donor HLA in renal allograft recipients. We have observed that PP/CMVIg durably eliminates antibody to donor HLA while antibodies to third party HLA antigens often recur or expand following the cessation of treatment. To understand the mechanism that underlies this apparent donor specific tolerance, we will characterize the effect PP/CMVIg treatments have on alloimmune anti-HLA-specific B and T cell responses in patients on our protocol. Evaluation of the immune function will be done at the following time points: 1) initiation of treatment; 2) time of transplant; 3) time of the last PP/CMVlg treatment; and 4) three months after completion of treatment. To analyze allo-specific B cells we have developed a novel, tetramer-based, approach to enumerate HLA-specific B cells. This assay will be used to assess the number of B cells specific for HLA class I antigens in sensitized patients receiving PP/CMVIg preconditioning due to a positive crossmatch with their HLA mismatched prospective donor. Data on the frequency of the various B cell populations, defined by HLA specificity, will be compared to the levels of circulating antibodies of comparable specificity and to the outcome of the transplant. AIIo-specific anti-HLA-A2 specific CTL response will also be analyzed in patients with antibody to HLA-A2. This will be done using classic anti-A2 specific analysis of CTL as well as using an artificial antigen-presenting cell (aAPC) we have also recently developed. Taken together, these studies will provide the first data on the apparent antigen specific tolerance that is induced by repeated PP/CMVIg treatments and will have implications for transplantation and possibly suggest novel approaches for the treatment of autoimmune disorders. [unreadable] [unreadable]